ERYTHROPOIETIN--STRUCTURE FUNCTION RELATIONSHIPS

促红细胞生成素--结构功能关系

• 批准号: 3486233
• 负责人: H. Franklin Bunn
• 金额: $ 36.76万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3486233
• 关键词: Escherichia coli; Insecta; X ray crystallography; acid base balance; antigens; biological signal transduction; calcium flux; cell free system; chemical structure function; computer simulation; erythropoietin; genetic manipulation; glycosylation; heme; laboratory mouse; membrane permeability; nucleic acid biosynthesis; phenylhydrazines; protein biosynthesis; protein structure function; radiotracer; receptor binding; site directed mutagenesis; tissue /cell culture; transfection; tritium

One of the major unanswered questions in the regulation of hematopoiesis is the mechanism by which soluble growth factors interact with specific receptors on hematopoietic cells. This proposal focuses on establishing the structural features of erythropoietin (Epo) that are responsible for its biosynthetic processing and function. Site-directed mutations, both deletions and amino acid replacements, will be introduced into the Epo gene in order to test specific functional properties. Methods will be developed for high level production of normal and mutant Epo's in Cos7, CHO and S. frugiperda cells as well as in E. Coli and in a cell-free translation system. The recombinant Epo products will be purified to homogeneity and characterized biochemically. the following assays will be developed to assess both short-term and long-term effects of normal and mutant products in the Epo-responsive cell line HCD57: equilibrium binding, internalization, Ca2+ influx, intracellular pH, [3H]-thymidine incorporation, and heme synthesis. For the latter 3 measurements, HCD57 cells will be compared to erythroid cells isolated from the spleens of mice treated with phenylhydrazine. We will prepare cassette mutants that correspond to surface epitopes predicted from primary and secondary structure. The properties of these mutants will provide information on the domains of Epo responsible for binding to its receptor as well as for internalization. Particular epitopes of interest will be selected for preparation of additional mutants that will better define these functionally important sites. Site-directed mutants will also be used to investigate the biosynthetic and functional roles of post- translational modifications including N-terminal processing, cleavage of C-terminal Arg, as well as N- and O- linked glycosylation. This cumulative information will be used in developing a computer based prediction of 3-dimensional structure. Concurrently we will attempt to prepare crystals of normal Epo and selected mutants, and if successful, they will be analyzed by x-ray diffraction. From these studies we hope to gain comprehensive information on structure-function relationships of Epo that will contribute to an understanding, at the molecular level, of receptor binding, internalization and signal transduction.

造血调节中尚未解答的主要问题之一 是可溶性生长因子与特定的相互作用的机制 造血细胞上的受体。 该提案的重点是建立 促红细胞生成素 (Epo) 的结构特征负责 其生物合成加工和功能。 定点突变，两者 删除和氨基酸替换，将被引入 EPO 基因以测试特定的功能特性。 方法将是 为 Cos7 中正常和突变 Epo 的高水平生产而开发， CHO 和草地贪夜蛾细胞以及大肠杆菌和无细胞 翻译系统。 重组Epo产品将被纯化为 均质性和生化特征。 以下测定将 开发用于评估正常情况的短期和长期影响 Epo 反应细胞系 HCD57 中的突变产物：平衡 结合、内化、Ca2+ 内流、细胞内 pH、[3H]-胸苷 掺入和血红素合成。 对于后 3 次测量，HCD57 细胞将与从脾脏分离的红细胞进行比较 用苯肼治疗的小鼠。 我们将准备盒式突变体 对应于从初级和次级预测的表面表位 结构。 这些突变体的特性将提供以下信息： 负责结合其受体的 Epo 结构域以及 为了内化。 将选择感兴趣的特定表位 用于制备额外的突变体，以更好地定义这些突变体 功能重要的站点。 定点突变体也将被使用 研究后的生物合成和功能作用 翻译修饰包括 N 端加工、裂解 C 末端精氨酸，以及 N- 和 O- 连接糖基化。 这 累积信息将用于开发基于计算机的 3维结构的预测。同时我们将尝试 制备正常 Epo 和选定突变体的晶体，如果成功， 它们将通过 X 射线衍射进行分析。 我们希望通过这些研究 获得有关结构与功能关系的全面信息 Epo 将有助于在分子水平上理解 受体结合、内化和信号转导。